Transplant tolerance, defined as indefinite allograft survival in the absence of immunosuppression and chronic rejection, has been achieved in monkeys and patients via infusion of recipients with donor bone marrow cells given in conjunction with kidney transplantation from the same living donor. Tolerance depends upon transient mixed hematopoietic chimerism and the activation of regulatory T cell. This procedure requires conditioning of recipients with donor bone marrow 6 days prior to transplantation and thus cannot be applied to recipients of organs from deceased donor. Recently, we have shown that tolerance can also be achieved via infusion of donor bone marrow cells performed several months after transplantation of monkeys with an allogeneic kidney whose survival had been maintained via conventional immunosuppression. However, this procedure called the delayed protocol is unsuccessful in over 50% of monkeys, which become sensitized to their donor owing to the generation and reactivation of donor-specific memory T cells after kidney transplantation (8-12). The limited success stresses the need to design novel strategies to harness alloreactive memory T cells while promoting the activation of regulatory T cells prior to or after transplantation, thereby facilitating tolerance. Recently, we have developed a mouse delayed mixed chimerism tolerance model, which recapitulates the features observed in monkeys and patients. These results correlate with previous observations in primates showing 1) the tolerogenic properties of delayed transient mixed chimerism, 2) the deleterious effects of donor-specific memory T cells, and 3) the requirement for regulatory B cells (Bregs) in allograft tolerance. Altogether, this suggests that treatments designed to stimulate Bregs or adoptive transfer of Bregs may be used to prevent or suppress donor-specific memory T cell responses and promote transplant tolerance induction in naive and sensitized transplant recipients. In this study, we propose the following specific aims: Specific Aim 1: To investigate the ability of regulatory B cells to inhibit the generation and reactivation of alloreactive memory T cells and to restore tolerance in nave and sensitized mice. Specific Aim 2: To investigate the mechanisms by which B cells control transplant tolerance induced via delayed mixed chimerism. If successful, these studies will provide insight into the mechanisms underlying tolerance achieved via delayed mixed chimerism and set the path for the design of B-cell-based strategies capable of harnessing alloreactive memory T cells, which is essential to the successful translation of our tolerance protocol in clinical settings.